Plasmid vector, method for detecting gene promoter activity, and assay kit

ABSTRACT

According to one embodiment, a first gene encodes a reporter protein. The first gene is disposed at the downstream of the gene promoter. A second gene is disposed at the downstream of the gene promoter and encodes a replication origin-binding protein. An internal ribosome entry site is disposed between the first gene and the second gene. The transcription termination signal sequence encodes a signal for terminating the transcription of the first gene and the second gene. A replication origin sequence is recognized by the replication origin-binding protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromJapanese Patent Application No. 2011-183896, filed Aug. 25, 2011, theentire contents of which are incorporated herein by reference.

FIELD

Embodiments described herein relate generally to a plasmid vector, amethod for detecting the activity of a gene promoter, and an assay kit.

BACKGROUND

Development of a novel method for the diagnosis of a disease based ongenome information has been expected since the base sequence of thehuman genome was determined. For example, genes relating to variousdiseases such as cancers have been found from gene analyses based on thebase sequence information of genomes. These disease-related genes anddisease-related proteins are attracting attentions as diagnosticbio-markers for diseases such as cancers. However, under the currentcircumstance, a stable result cannot be obtained by a diagnosis bydetecting a bio-marker, for the reasons that sufficient expression ofthe bio-marker cannot be necessarily obtained at an early stage of adisease, and the like.

Early diagnosis of diseases is essential for the future medicaltreatments from the viewpoints of improvement in cure rates andreduction of the burden by patients. As an approach for early diagnosis,a method comprising detecting the change in the promoter activity of agene that expresses at an early stage of a disease. The probability ofexpression of the gene is controlled by the change in the activity of agene promoter. The activity of gene promoters is observed earlier thanthe expression of disease-related genes and disease-related proteins.

As a method for detecting the activity of a gene promoter in a cell, areporter gene assay may be exemplified. The reporter gene assay is amethod comprising introducing a reporter vector in which an expressioncassette to which a reporter gene for visualizing the activity of apromoter is bound is incorporated at the downstream of the promoter intoa subject cell, and quantifying the activity of the promoter based onthe activity of a reporter protein. As the reporter gene, a luciferasegene, a β-galactosidase gene, a fluorescent protein gene and the likeare adopted.

However, for example, at an early stage of a disease, the number of cellin a pre-disease state by the change in the activity of a gene promoteris quite little. It cannot be considered that the probability ofincorporation of the cell in blood or a tissue collected for diagnosisis high. Therefore, a technique for detection with high sensitivity isrequired for early diagnosis of a disease and the like.

At present, as a reporter gene assay with high sensitivity, a method bya virus reporter vector in which a gene promoter and a fluorescentprotein are incorporated in a genome of a virus has been reported. Inthis reporter gene assay, the sensitivity of the reporter gene assay isincreased by increasing the amount of expression of the fluorescentprotein by proliferating a virus vector in a host cell infected with thevirus vector, thereby increasing the amount of expression of thefluorescent protein. However, a virus reporter vector utilizes theability of infection of a virus for introducing the vector into a hostcell. Therefore, there is a risk of infection of an operator with thevirus reporter vector. Accordingly, the handling of the virus reportervector is not convenient, and there is a problem in safeness.

On the other hand, a plasmid reporter vector is handled moreconveniently than the virus reporter vector is, and has no infectivenessand has high safeness. Many reporter gene assays using a plasmidreporter vector have been reported. An application of a plasmid reportervector to early diagnosis of diseases is expected. However, unlike avirus reporter vector, a plasmid vector does not proliferate in a hostcell, and thus there is a problem in the detection sensitivity thereof.

The object of the embodiment is to provide a plasmid vector, a methodfor detecting a gene promoter and an assay kit, by which the activity ofa gene promoter can be detected with high sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing that schematically shows the structure of theplasmid vector according to the present embodiment;

FIG. 2 is a drawing that shows the whole image of a method for detectingthe activity of a gene promoter (a reporter gene assay) using theplasmid vector of FIG. 1;

FIG. 3 is a drawing that schematically shows a typical flow of themethod for preparing the plasmid vector according to the presentembodiment pCMV-Luc-IRES-LT;

FIG. 4A is a drawing that schematically shows the structure of thepCMV-Luc-IRES-LT according to an example of the present embodiment;

FIG. 4B is a drawing that schematically shows the structure of thepCMV-Luc-IRES-LT-E107K according to an example of the presentembodiment;

FIG. 4C is a drawing that schematically shows the structure of thepCMV-Luc-IRES-LT-D402E according to an example of the presentembodiment;

FIG. 5 is a drawing that schematically shows a typical flow of a methodfor preparing the vector pCMV-Luc for a negative control according tothe present embodiment;

FIG. 6 is a drawing that shows the result of the detection of theamounts of expression of an LT protein in the pCMV-Luc-IRES-LT andpCMV-Luc according to examples of the present embodiment;

FIG. 7A is a drawing showing the result of the comparison of the copynumbers of the pCMV-Luc-IRES-LT and pCMV-Luc according to examples ofthe present embodiment, which shows an ethidium bromide-stained pictureof the pCMV-Luc-IRES-LT and pCMV-Luc;

FIG. 7B is a drawing showing the result of the comparison of the copynumbers of the pCMV-Luc-IRES-LT and pCMV-Luc according to examples ofthe present embodiment, which shows a graph (a graph of a stainingintensity ratio of the bands in FIG. 7A) for the comparison of the copynumbers of the pCMV-Luc-IRES-LT and pCMV-Luc;

FIG. 8 is a drawing that shows the results of the detection of the lightemission signals of the reporter protein in the pCMV-Luc-IRES-LT andpCMV-Luc according to examples of the present embodiment;

FIG. 9 is a drawing that shows the result of the comparison of thesignal intensities of the reporter protein in the pCMV-Luc-IRES-LT,pCMV-Luc-IRES-LT-E107K and pCMV-Luc-IRES-LT-D402E according to examplesof the present embodiment with the pCMV-Luc; and

FIG. 10 is a drawing that shows the whole image of the method fordetecting the activity of a gene promoter using a non-amplified reportervector according to a conventional example.

DETAILED DESCRIPTION

In general, according to one embodiment, a plasmid vector includes agene promoter, a first gene, a second gene, an internal ribosome entrysite, a transcription termination signal sequence and a replicationorigin sequence. The first gene encodes a reporter protein forvisualizing the activity of the gene promoter which is disposed at thedownstream of the gene promoter. The second gene is disposed at thedownstream of the gene promoter and encodes a replication origin-bindingprotein. The internal ribosome entry site is disposed between the firstgene and the second gene. The transcription termination signal sequenceencodes a signal for terminating the transcription of the first gene andthe second gene. The replication origin sequence is recognized by thereplication origin-binding protein.

Hereinafter the plasmid vector, the method for detecting a genepromoter, and the assay kit according to the present embodiment will beexplained with referring to drawings.

The plasmid vector according to the present embodiment is a plasmid-typereporter vector that enhances a detection signal by the specificself-amplification of a gene promoter to be detected in an activatedhost cell.

[Structure of Plasmid Vector]

FIG. 1 is a drawing that schematically shows the structure of theplasmid vector 1 that is a self-amplified reporter vector according tothe present embodiment. As shown in FIG. 1, the plasmid vector 1according to the present embodiment has a gene promoter 10 to bedetected. A base sequence 20 for transcription and translation isdisposed at the downstream of the gene promoter 10 to be detected. Thebase sequence 20 includes a reporter gene 21, an internal ribosome entrysite (IRES) 23, a replication origin-binding protein gene 25 and atranscription termination signal sequence 27. The IRES 23 is disposedbetween the reporter gene 21 and the replication origin-binding proteingene 25. The transcription termination signal sequence 27 is disposed atthe downstream of the replication origin-binding protein 25.Furthermore, the plasmid vector 1 has a replication origin sequence 30at the sites other than the site between the gene promoter 10 and thetranscription termination signal sequence 27 at the side that thereporter gene 21 and the replication origin-binding protein gene 25 gothrough. Although it is explained that the reporter gene 21 is disposedat the upper stream of the IRES 23 and the replication origin-bindingprotein gene 25 is disposed at the downstream, the present embodiment isnot limited to this. For example, the replication origin-binding proteingene 25 may be disposed at the upper stream of the IRES 23 and thereporter gene 21 may be disposed at the downstream. In this case, it ispreferable that the transcription termination signal sequence 27 isdisposed at the downstream of the reporter gene 21.

Hereinafter the individual elements included in the plasmid vector 1will be explained in detail.

The gene promoter 10 to be detected is a base sequence having a bindingsequence of an RNA polymerase. The gene promoter 10 activates thetranscription of a gene that is functionally bound to a binding sequenceof an RNA polymerase. An arbitrary gene promoter may be used for thegene promoter 10 depending on the intended purpose. For example, ifearly diagnosis of a disease is intended, an arbitrary disease-relatedgene promoter that has been activated at the early stage of the diseasecan be used as the gene promoter 10. Specifically, in the case when thedisease is a cancer, gene promoters such as c-fos, c-myc, CD13, CD44,CD90, Snail and drug resistant transporter genes (ABCG, MDR), and thelike can be utilized as the gene promoter 10. If detection of anenvironmental stimulus such as harmful chemical substances, temperaturesand oxidation stress is intended, a gene promoter that is activated inresponse to the intended environmental stimulus can be used as the genepromoter 10. Specifically, in the case when the environmental stimulusis a harmful chemical substance, a gene promoter of cytochrome p450 orthe like can be utilized as the gene promoter 10. In addition, the genepromoter 10 may include an arbitrary enhancer. An enhancer is a basesequence that is linked to the gene promoter 10. The enhancer has afunction of enhancing the activation of the transcription of a gene bythe gene promoter 10.

The reporter gene 21 is a gene (base sequence) that encodes a reporterprotein. The reporter gene 21 has a role to visualize the activity ofthe gene promoter 10. As the reporter gene 21, all of reporter genesthat are already known in the art can be applied. As the reporter gene21, a reporter gene that can easily measure the activity of a reporterprotein that is a translation product of the reporter gene and providesa low measurement background is preferable. Specific examples of thereporter gene 21 may include a luminescent enzyme gene, a fluorescentprotein gene, a color-developing enzyme gene, an activeoxygen-generating enzyme gene, a heavy metal-binding protein gene andthe like. The reporter gene 21 can be suitably selected depending on anapparatus for detecting a reporter protein.

The replication origin-binding protein gene 25 is a gene that encodes areplication origin-binding protein. The replication origin-bindingprotein is a protein that may function to origin the replication of aDNA in a host cell. The replication origin-binding protein may be, forexample, a protein derived from a virus that is amplified by infectionwith a host cell, or a protein of the same animal species as that of ahost cell or a protein of a different animal species from that of a hostcell.

The IRES 23 is a base sequence for transcripting the reporter gene 21and the replication origin-binding protein gene 25 to a single mRMA (abicistronic mRMA) so that a reporter protein that is a translationproduct of the reporter gene 21 and a replication origin-binding proteinthat is a translation product of the replication origin-binding proteingene 25 can be synthesized individually. More specifically, the IRES 23is a base sequence that initiates the translation of a protein bybinding a ribosome to the inside of an mRNA without depending on the5′-end cap structure of the mRNA in a mammal cell. By disposing the IRES23 between the two genes, a bicistronic mRNA is synthesized. Namely, byincorporating the IRES 23 between the two genes, two kinds of proteinsthat are encoded by these two genes are translated in one mRNA. Examplesof the IRES 23 may include an IRES of encephalomyocarditis virus (ECMV),and the like. The IRES 23 according to the present embodiment isdisposed between the reporter gene 21 and the replication origin-bindingprotein gene 25 to link the reporter gene 21 to the replicationorigin-binding protein gene 25. Therefore, a reporter protein and areplication origin-binding protein are translated from one bicistronicmRNA.

The transcription termination signal sequence 27 is a base sequence thatencodes a signal for terminating the transcription of the reporter gene21 and the replication origin-binding protein gene 25 at the upperstream. The transcription termination signal sequence 27 may be one thatfunctions to terminate the transcription of a gene of a mammal. Examplesof the transcription termination signal sequence 27 may include a latepoly (A) addition signal sequence of Simian Virus 40 (SV40), a poly (A)addition signal sequence of bovine growth hormone gene, and the like.However, the transcription termination signal sequence 27 according tothe present embodiment is not limited to these signal sequences. As longas the functions of a transcription termination signal sequence are notimpaired, these signal sequences whose gene sequences have been modifiedmay also be used as the transcription termination signal sequence 27.

A replication origin sequence 30 is a base sequence that can berecognized by a replication origin-binding protein that is synthesizedby the expression of the replication origin-binding protein gene 25. Thereplication of the plasmid vector 1 is initiated by the recognition ofand binding to the replication origin sequence 30 by the replicationorigin-binding protein. The replication origin sequence 30 may be onederived from the same origin as that of the animal species of thereplication origin-binding protein, or may be one derived from adifferent origin.

For example, in the case when the plasmid vector 1 is introduced into acell of a primate such as a human and a simian, a large T antigen (LT)gene of SV40 can be used as the replication origin-binding protein gene25, and an on sequence of SV40 can be used as the replication originsequence 30. Alternatively, in the case when the plasmid vector 1 isintroduced into a cell of a primate such as a human and a simian, anEBNA-1 protein of Epstein-Barr virus (EBV) can be used as thereplication origin-binding protein gene 25, and an EB virus latentorigin of replication (oriP) can be used as the replication originsequence 30. Furthermore, in the case when the plasmid vector 1 isintroduced into a cell of a rodent such as a mouse, a large T antigen(LT) gene of Mouse polyomavirus (PyV) can be used as the replicationorigin-binding protein gene 25, and a PyV core origin sequence can beused as the replication origin sequence 30.

As mentioned above, the plasmid vector 1 according to the presentembodiment comprises the reporter gene 21, the replicationorigin-binding protein 25 and the replication origin sequence 30 in thesame vector. By doing so, the plasmid vector 1 according to the presentembodiment functions as a self-amplified reporter vector.

[Detection of Activity of Gene Promoter]

The method for detecting the activity of the gene promoter 10 accordingto the present embodiment (a reporter gene assay) detects the activityof the gene promoter 10 to be detected by using the plasmid vector (aself-amplified reporter vector) 1 according to the present embodiment.

FIG. 2 is a drawing that shows the whole image of a reporter gene assayusing the plasmid vector according to the present embodiment.

As shown in FIG. 2, the plasmid vector 1 is first introduced into a hostcell. As the method for introducing the plasmid vector 1 into the hostcell, an already-known cell engineering technique is used. For example,as the method for introducing the plasmid vector 1 into the host cell, abiochemical method or a physicochemical method is used. Examples of thebiochemical method may include a lipofection method using a cationlipid, a magnetofection method using magnetic particles, and a methodusing calcium chloride, and the like. Examples of the physicochemicalmethod may include an electroporation method, a sonoporation method, andthe like. As the method for introducing the plasmid vector 1 into thehost cell, the above-mentioned biochemical methods and physicochemicalmethods may be used alone or in combination. For example, thelipofection method and magnetofection method which are biochemicalmethods may be combined, or the lipofection that is a biochemical methodand an electroporation method that is a physicochemical method may becombined.

After the plasmid vector 1 has been introduced into the host cell, thehost cell is cultured over an arbitrary period under a culture conditionin which the host cell can divide and proliferate. This culturecondition is a condition in which the plasmid vector 1 can be amplified.In this culture period, in the case when the state of the host cell orthe environment in which the host cell is placed satisfies theactivation condition that is inherent to the gene promoter 10, the genepromoter 10 is activated. Thus, in the case when the gene promoter 10 isin an activated state in a host cell that satisfies the activationcondition (target cell), the reporter gene 21 and the replicationorigin-binding protein gene 25 which have been linked in the IRES 23 aretranscripted to a bicistronic mRNA and translated. By thesetranscription and translation, a reporter protein (a signal to bemeasured) and a replication origin-binding protein are synthesized. Thereplication origin-binding protein recognizes and binds the replicationorigin sequence 30 and recruits plural proteins relating to thereplication of DNAs included in the host cell (a DNA replicationapparatus). The plasmid vector 1 is replicated in the host cell by therecruited plural proteins. By this way, the plasmid vector 1 repeatsreplication one after another in the host cell and amplifies a copynumber. In accordance with the amplification of a copy number, thenumber of reporter proteins synthesized by the plasmid vector 1 is alsoincreased.

On the other hand, in the case when the host cell does not satisfy theactivation condition of the gene promoter 10, the gene promoter 10 isnot activated. In a host cell that does not satisfy the activationcondition (a non-targeted cell), the reporter gene 21 and thereplication origin-binding protein gene 25 are not transcripted andtranslated. Therefore, a reporter protein and a replicationorigin-binding protein are not synthesized. In this case, a measurementsignal is not generated in the non-targeted cell. Since a replicationorigin-binding protein is not synthesized, the plasmid vector 1 cannotconduct replication.

By this way, the plasmid vector 1 according to the present embodiment isself-amplified only in a host cell (target cell) that satisfies theactivation condition of the gene promoter 10. Next, a conventionalplasmid vector (non-amplified reporter vector) shown in FIG. 10 will beconsidered. A conventional plasmid vector does not have a replicationorigin-binding protein and a replication origin sequence, and thuscannot be self-amplified even in a target cell. Therefore, the plasmidvector 1 according to the present embodiment can increase the signal ofa reporter protein in a target cell, i.e., an object to be measured, ascompared to the conventional plasmid vector. Therefore, the sensitivityof the reporter gene assay can be increased.

Hereinafter the detection of the amount of expression of the reporterprotein will be explained in detail. As mentioned above, a luminescentenzyme gene, a fluorescent protein gene, a color-developing enzyme gene,an active oxygen-generating enzyme gene, a heavy metal-binding proteingene or the like can be selected as the reporter gene 21.

The luminescent enzyme gene is a gene that encodes an enzyme proteinthat catalyzes a luminescent reaction. Examples of the luminescentenzyme gene may include a luciferase gene. Luciferase is a translationproduct of the luciferase gene. Luciferase conducts a luminescentreaction by using luciferin that is one of the substrates thereof.

The fluorescent protein gene is a gene that encodes a fluorescentprotein. Examples of the fluorescent protein gene may include a bluefluorescent protein gene, a green fluorescent protein gene and a redfluorescent protein gene. The blue fluorescent protein that is atranslation product of the blue fluorescent protein gene emits bluefluorescence. The green fluorescent protein that is a translationproduct of the green fluorescent protein gene emits green fluorescence.The red fluorescent protein that is a translation product of the redfluorescent protein gene emits red fluorescence.

The color-developing enzyme gene is a gene that encodes an enzymeprotein that catalyzes a color-development reaction. Examples of thecolor-developing enzyme gene may include a β-galactosidase gene.β-galactosidase that is a translation product of the β-galactosidasegene conducts a color development reaction by using a substrate such as5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) ando-nitrophenyl-β-D-galactopyranoside (ONPG).

The translation products of the luminescent enzyme gene, fluorescentprotein gene and color-developing enzyme gene can be detected by anoptical detection apparatus. Specifically, in the case when the reportergene 21 is a luciferase gene, a luciferase protein is extracted from ahost cell, and a substrate is added to the extracted luciferase protein.The luminescence intensity of the solution comprising the luciferaseprotein and substrate is measured by a luminometer. In the case when thereporter gene is a fluorescent protein gene, for example, a host cell isirradiated with laser. By the irradiation of laser, fluorescence isgenerated from the fluorescent protein in the host cell. The intensityof the generated fluorescence is measured by a fluorophotometer.Alternatively, in the case when the reporter gene is a fluorescentprotein gene, the intensity of fluorescence may be measured as follows.First, an extraction liquid comprising the fluorescent protein extractedfrom the host cell is irradiated with laser. By the irradiation oflaser, fluorescence is generated from the fluorescent protein in theextraction liquid. The intensity of the fluorescence is measured by afluorophotometer. In the case when the reporter gene is aβ-galactosidase gene, a β-galactosidase protein is extracted, and asubstrate is added to the extracted β-galactosidase protein. Theabsorbance of the solution comprising the β-galactosidase protein andsubstrate is measured by an absorbance measurement apparatus.

The active oxygen-generating enzyme gene is a gene that encodes anenzyme that generates active oxygen. Examples of the activeoxygen-generating enzyme gene may include a nitric monoxide synthasegene, a xanthine oxidase gene and the like. Nitric monoxide synthasethat is a translation product of the nitric monoxide synthase gene andxanthine oxidase that is a translation product of the xanthine oxidasegene generate active oxygen.

Active oxygen can be detected by an electron spin resonance (ESR)apparatus. In a method by the ESR apparatus, the amount of generation ofactive oxygen may be measured directly, or may be measured by utilizinga specific spin trap agent. In the case when the spin trap agent isutilized, at first, an active oxygen-generating enzyme is trapped by thespin trap agent. Then, the amount of generation of the active oxygengenerated from the trapped active oxygen-generating enzyme is measuredby the ESR apparatus.

The heavy metal-binding protein gene is a gene that encodes a proteinthat specifically binds to a heavy metal. The amount of generation ofthe protein bound to the heavy metal can be measured by a diagnosticimaging apparatus such as a magnetic resonance imaging (MRI) apparatus,a positron emission computed tomography (PET) apparatus and a computedtomography (CT) apparatus. For example, the amount of generation of theheavy-metal-binding protein is conducted as follows. First, a heavymetal that can be measured is added to a culture liquid of a host cell.The host cell is then washed, and a heavy-metal-binding protein isextracted from the host cell. An image of the extractedheavy-metal-binding protein is then photographed by an image diagnosisapparatus that corresponds to the heavy metal added to the cultureliquid. The image diagnosis apparatus generates an image relating to theheavy-metal-binding protein. In the image diagnosis apparatus, thegenerated image is subjected to an image processing and the amount ofthe heavy-metal-binding protein is measured. The heavy-metal-bindingprotein may be expressed inside of the host cell, or may be expressed onthe outer surface of the host cell.

In the case when a gene that encodes a protein that binds aferromagnetic metal such as iron, gadolinium or a complex compoundthereof is utilized as the heavy metal-binding protein gene, a nuclearmagnetic resonance apparatus such as an MRI apparatus is preferable asthe image diagnosis apparatus. In this case, the heavy metal-bindingprotein gene may be an antibody gene or single-strand antibody gene thatspecifically binds to a ferromagnetic metal such as the above-mentionedcomplex compounds. Specifically, as the heavy metal-binding proteingene, ferritin and transferrin which are iron-binding proteins, orsingle-strand antibodies having an ability of binding to gadolinium aresuitable.

In the case when a gene that encodes a protein that binds a radioactiveisotope metal such as strontium, copper, technetium, gallium or complexcompounds thereof is utilized as the heavy metal-binding protein gene, aradiation measurement apparatus such as a PET apparatus and a CTapparatus is preferable as the image diagnosis apparatus. In this case,an antibody gene or single-strand antibody gene that specifically bindsto a radioactive isotope metal such as the above-mentioned complexcompounds may be used as the heavy metal-binding protein gene.

Thus, according to the reporter gene assay according to the presentembodiment, the amount of expression of the reporter protein from theplasmid vector 1 can be measured by a detection apparatus correspondingto the reporter protein. Furthermore, the presence or absence of theactivity of the gene promoter 10 can be detected by the comparison ofthe measured value and threshold value of the amount of expression byutilizing a detection apparatus. For example, in the case when themeasured value of the amount of expression is larger than a thresholdvalue, it is considered that the activity of the gene promoter 10 isdetected, and in the case when the measured value of the amount ofexpression is smaller than the threshold value, it may be consideredthat the activity of the gene promoter 10 is not detected. The thresholdvalue can optionally be set by a user depending on the gene promoter 10,the reporter gene 21, the host cell and the like.

As mentioned above, the gene promoter 10 can be suitably selecteddepending on an object to be detected. For example, in the case ofdiagnosis of a cancer, it is preferable that a gene promoter of acancer-related gene such as c-fos, c-myc, CD13, CD44, CD90, Snail anddrug-resistant transporter genes (ABCG, MDR) is used as the genepromoter 10. By doing so, the plasmid vector 1 is amplified in a cancercell and the amount of expression of the reporter protein is increased,and thus the cancer cell included in the host cell can be detected withhigh sensitivity. As another example, in the case of detection of anenvironmental stimulus, it is preferable to use a environment-specificpromoter as the gene promoter 10. Therefore, by providing a desiredenvironmental stimulus such as a chemical substance, a temperature andan oxidation stress to the host cell, the plasmid vector 1 is amplifiedin the cell and the amount of expression of the reporter protein isincreased, and thus the environmental stimulus can be detected with highsensitivity.

Furthermore, as a cell that can be used as the host cell, cells derivedfrom primates including human and simians, rodents such as mice andrats, and the like can be applied. Furthermore, established cell linesand primacy cultured cells can also be applied as these cells. Examplesof established cell lines that are derived from primates may includeHuh-7 and HepG2 derived from human hepatic cancer, Jurkat and HL60derived from human blood corpuscle cells, MCF-7 derived from humanbreast cancer and CV-1 derived from simian kidney, or subclones andgenetically modified cells thereof, and the like. Examples ofestablished cell lines derived from rodents may include Hepa-1 derivedfrom murine hepatic cancer, Neuro2a derived from murine neuroblastoma,PC12 derived from rat pheochromocytoma, or subclones and geneticallymodified cells thereof, and the like. Examples of the primacy culturedcells may include cells and tissue stem cells which are derived fromorgans of the digestive system including liver, lungs, kidneys and thelike, the circulation system including leucocytes and the like, theendocrine system including thyroid gland and the like, or the centralnerve system including brain, spinal cord and the like, and the like.

The host cell used in the reporter gene assay according to the presentembodiment may be selected depending on the kinds of the replicationorigin-binding protein gene 25 and replication origin sequence 30 whichare incorporated into the plasmid vector 1. For example, in the casewhen the replication origin-binding protein gene 25 is an LT proteingene of SV40 and the replication origin sequence 30 is SV40ori, or inthe case when the replication origin-binding protein gene 25 is anEBNA-1 protein of EBV and the replication origin sequence 30 is oriP ofEBV, it is preferable to select a cell derived from a primate such as ahuman and a simian as the host cell. Furthermore, in the case when thereplication origin-binding protein gene 25 is an LT protein of PyV andthe replication origin sequence 30 is a PyV core origin sequence, it ispreferable to select a cell derived from a rodent such as a mouse as thehost cell.

The kind of the cell is not specifically limited as long as thecombination of the plasmid vector 1 and the animal species of the hostcell is correct. For example, the host cell may also be a establishedcell line or a primary cultured cell. Furthermore, the tissue from whichthe host cell is derived is also not specifically limited. This may beselected depending on the kind of the gene promoter 10 to be detected.

Thus, by using the plasmid vector 1 according to the present embodiment,a method for detecting the activity of the gene promoter 10 with highsensitivity, i.e., a highly-sensitive reporter gene assay can beprovided.

[Assay Kit]

The assay kit according to the present embodiment is used in thereporter gene assay according to the present embodiment.

The assay kit according to the present embodiment has the plasmid vector1 according to the present embodiment and a detection reagent. Thedetection reagent is a reagent for detecting the expression of thereporter gene 21 that encodes the reporter protein included in theplasmid vector 1 in the cell. Furthermore, the assay kit according tothe present embodiment may further have an introduction reagent and anextraction reagent. The introduction reagent is a reagent forintroducing the plasmid vector 1 into a cell. The extraction reagent isa reagent for extracting the reporter protein from the cell.Furthermore, the assay kit according to the present embodiment mayinclude a reaction cuvette and a cell culture vessel. The reactioncuvette is a cuvette for conducting a reaction by the detection reagent,introduction reagent and extraction reagent. The cell culture vessel isa vessel for culturing the host cell to which the plasmid vector 1 hasbeen introduced. Furthermore, the assay kit according to the presentembodiment may further comprise a culture medium for culturing and abuffer.

Thus, the assay kit according to the present embodiment can detect theactivity of the gene promoter 10 with high sensitivity by being used inthe reporter gene assay according to the present embodiment.

EXAMPLES

1. Base Sequence of Plasmid Vector

Next, the plasmid vector according to the present embodiment will beexplained with referring to cases when a cell of a primate such as ahuman and a simian is used as a host cell as specific examples. As theplasmid vector 1 in these specific examples, pCMV-Luc-IRES-LT isadopted. An LT gene of SV40 is adopted as a replication origin-bindingprotein gene 25 for this pCMV-Luc-IRES-LT, and a base sequence that canbe recognized by an LT protein that is a translation product of the LTgene is adopted as the replication origin sequence 30. Furthermore, apromoter of cytomegalovirus is adopted as a gene promoter 10 ofpCMV-Luc-IRES-LT, a luciferase gene of a photinus pyralis (firefly) isadopted as a reporter gene 21, an IRES of encephalomyocarditis virus isadopted as an IRES 23, and a transcription termination signal sequenceof bovine growth hormone gene is adopted as a transcription terminationsignal sequence 27.

The base sequence of the LT gene of SV40 is shown in SEQ ID NO: 1 (wildtype) of the Sequence Listing. The base sequence of the LT gene does notneed to be completely identical with the base sequence of SEQ ID NO: 1as long as the function of an LT protein to initiate DNA replication isnot lost. For example, a mutant-type LT gene in which an arbitrarymutation that has a possibility to enhance a function of initiating DNAreplication has been introduced into the base sequence of an LT gene maybe prepared and incorporated into the plasmid vector 1. Examples of thebase sequence of such mutant-type LT gene are shown in SEQ ID NO: 2(mutant-type 1), SEQ ID NO: 3 (mutant-type 2) and SEQ ID NO: 4(mutant-type 3) in the Sequence Listing. The mutant-type LT geneaccording to SEQ ID NO: 2 is different from the LT gene according to SEQID NO: 1 in that the 319-th base guanine (G) has been substituted withadenine (A), and the 321-th base adenine (A) has been substituted withguanine (G). By this substitution of the bases, an LT protein that is atranslation product of the LT gene according to SEQ ID NO: 2 isdifferent from an LT protein of a translation product of the LT geneaccording to SEQ ID NO: 1 in that the 107-th amino acid glutamic acidhas been substituted with lysine. The mutation is an example of amutation in which the interaction between an LT protein and an Rbprotein (a protein having a function of suppressing canceration) withoutimpairing the DNA replication function of the LT protein. Themutant-type LT gene according to SEQ ID NO: 3 is different from the LTgene according to SEQ ID NO: 1 in that the 1206-th base thymine (T) hasbeen substituted with guanine (G). By this substitution of the base, anLT protein that is a translation product of the LT gene according to SEQID NO: 3 is different from an LT protein that is a translation productof the LT gene according to SEQ ID NO: 1 in that the 402-th amino acidaspartic acid has been substituted with glutamic acid. The mutation isan example of mutation in which the interaction between an LT proteinand a p53 protein (a protein having a function of suppressingcanceration) is inhibited without impairing the DNA replication functionof the LT protein. SEQ ID NO: 4 is a mutant-type LT gene that wasprepared by introducing both the substitution of SEQ ID NO: 2 and thesubstitution of SEQ ID NO: 3 into the LT gene according to SEQ ID NO: 1.The above-mentioned LT genes are only examples, and the kinds and sitesof the base and amino acid sequence to be substituted do not need to beidentical with those of the mutant-type LT genes shown herein as long asthe mutation has a possibility of enhancing a function of initiating DNAreplication.

In the case when the replication origin-binding protein gene 25 is an LTgene of SV40, for example, an on sequence of SV40 is suitable as thereplication origin sequence 30. An example of the on sequence of SV40 isshown in SEQ ID NO: 5 of the Sequence Listing. This base sequence alsodoes not need to be completely identical with the base sequence of SEQID NO: 5 as long as the function to initiate replication is not lost.

The base sequences of the LT gene, mutant-type LT gene, the on sequenceof SV40 and the like can be acquired by known genetic engineeringtechniques. For example, it is possible to amplify and acquire a DNAcomprising a base sequence to be acquired by PCR (polymerase chainreaction) utilizing a primer set that is specific to these basesequences to be acquired. Alternatively, all of the base sequences to beacquired may be synthesized artificially. Alternatively, a base sequencethat has been already incorporated in a vector may be utilized.

An example of a promoter of cytomegalovirus which is the gene promoter10 is shown in SEQ ID NO: 6 in the Sequence Listing, an example of aluciferase gene of a photinus pyralis which is the reporter gene 21 isshown in SEQ ID NO: 7 in the Sequence Listing, an example of an IRES ofencephalomyocarditis virus is shown in SEQ ID NO: 8 in the SequenceListing, and an example of a transcription termination signal sequenceof bos Taurus (bovine growth hormone) gene is shown in SEQ ID NO: 9 inthe Sequence Listing. The plasmid vector 1 according to the presentembodiment is not limited by these genes and base sequences.Furthermore, the base sequences of the genes and signals do not need tobe identical with the base sequences described in the above-mentionedSEQ ID NOS as long as their functions are not lost.

2. Preparation of Vector

Next, a method for preparing the plasmid vector pCMV-Luc-IRES-LT will beexplained. FIG. 3 is a drawing that schematically shows a typical flowof a method for preparing the plasmid vector pCMV-Luc-IRES-LT.

As shown in FIG. 3, a vector PGV-B2(TOYO B-Net), a vector pBSII-IRES anda vector pCMV-LT (w/oZeo) are prepared. The vector PGV-B2 includes aluciferase gene 41 of a firefly. The vector pBSII-IRES includes an IRES43 of encephalomyocarditis virus. The vector pCMV-LT includes a genepromoter of CMV (cytomegalovirus) 45, an LT gene 47, a transcriptiontermination signal sequence of bovine growth hormone gene 49, and anSV40 on sequence 51. The gene promoter of cytomegalovirus 45 is referredto as a CMV promoter hereinafter.

First, the PGV-B2 is digested by a restriction enzyme SacI and arestriction enzyme XbaI to cut out the luciferase gene 41. Similarly,the pBSII-IRES is cut by a restriction enzyme SacI and a restrictionenzyme XbaI. Furthermore, the luciferase gene 41 that has been cut outfrom the PGV-B2 is linked to the pBSII-IRES that has been cut by therestriction enzyme SacI and the restriction enzyme XbaI by T4 ligase toprepare pBSII-Luc-IRES.

The pBSII-Luc-IRES is then cut by a restriction enzyme PstI. The3′-protruding end of the pBSII-Luc-IRES that has been cut by therestriction enzyme PstI is smoothen by T4DNA polymerase. After thesmoothing, a DNA fragment comprising the luciferase gene 41 and the IRES43 is cut out from the pBSII-Luc-IRES by cutting the pBSII-Luc-IRES by arestriction enzyme NheI. Meanwhile, the pCMV-LT is also subjected to asimilar treatment to that the pBSII-Luc-IRES has undergone. Namely, thepCMV-LT is cut by a restriction enzyme PstI, the 3′-protruding end issmoothen by T4DNA polymerase, and the pCMV-LT is cut by a restrictionenzyme NheI. Furthermore, a DNA fragment that has been cut out from thepBSII-Luc-IRES is linked to the pCMV-LT by T4 ligase, thereby a plasmidvector pCMV-Luc-IRES-LT according to the present embodiment is prepared.By the above-mentioned treatments, the pCMV-Luc-IRES-LT comprises theCMV promoter 45, luciferase gene 41, IRES 43, LT gene 47, transcriptiontermination signal sequence 49 and replication origin sequence 51. Theluciferase gene 41, IRES 43, LT gene 47 and transcription terminationsignal sequence 49 are incorporated at the downstream of the CMVpromoter 45.

Furthermore, pCMV-Luc-IRES-LT-E107K and pCMV-Luc-IRES-LT-D402E in whichthe LT gene of the above-mentioned plasmid vector pCMV-Luc-IRES-LT wasrecombined to form a mutant-type LT gene were prepared as other plasmidvectors 1. FIG. 4A is a drawing that schematically shows the structureof the pCMV-Luc-IRES-LT, FIG. 4B is a drawing that schematically showsthe structure of the pCMV-Luc-IRES-LT-E107K, and FIG. 4C is a drawingthat schematically shows the structure of the pCMV-Luc-IRES-LT-D402E. Asshown in FIG. 4A, the 319-th base of the LT gene 47 of thepCMV-Luc-IRES-LT is guanine (G), the 321-th base is adenine (A), and the1206-th base is thymine (T). As shown in FIG. 4B, the 319-th base of theLT gene 47′ of the pCMV-Luc-IRES-LT-E107K is adenine (A), the 321-thbase is guanine (G), and the 1206-th base is thymine (T). As shown inFIG. 4C, the 319-th base of the LT gene 47″ of thepCMV-Luc-IRES-LT-D402E is guanine (G), the 321-th base is adenine (A),and the 1206-th base is guanine (G). The pCMV-Luc-IRES-LT-E107K isprepared by a site-directed mutagenesis method of a plasmid bysubstituting the 319-th base guanine (G) with adenine (A) and the 321-thbase adenine (A) with guanine (G) in the LT gene of thepCMV-Luc-IRES-LT. The pCMV-Luc-IRES-LT-D402E is prepared by asite-directed mutagenesis method of a plasmid by substituting the1206-th base thymine (T) with guanine (G) in the LT gene of thepCMV-Luc-IRES-LT.

This is the end of the explanation on the specific example of the methodfor preparing the plasmid vector 1 according to the present embodiment.

pCMV-Luc was prepared as a vector for a negative control to beintroduced into a host cell together with the plasmid vector 1 or theplasmid vector 1 that has been recombined to a mutant-type LT gene. FIG.5 is a drawing that schematically shows a typical flow of the method forpreparing the vector pCMV-Luc. As shown in FIG. 5, the pCMV-Luc wasprepared from pCMV-LT (w/oZeo) and a vector PGV-B2. The pCMV-LTcomprises a CMV promoter 61, an LT gene 63, a transcription terminationsignal sequence 65 and an SV40 on sequence 67. The PGV-B2 comprises aluciferase gene 69. The pCMV-Luc can be prepared by recombining the LTgene 63 of the pCMV-LT and the luciferase gene 69 of the PGV-B2.

Specifically, at first, the PGV-B2 is cut by a restriction enzymeHindIII and a restriction enzyme XbaI to cut out the luciferase gene 69.Similarly, the pCMV-LT is cut out by the restriction enzyme HindIII andthe restriction enzyme XbaI to remove the LT gene 63 from the pCMV-LT.Furthermore, a vector pCMV-Luc for a negative control is prepared bylinking the luciferase gene 69 that has been cut out, to the pCMV-LTthat has been cut by the restriction enzyme HindIII and the restrictionenzyme XbaI, by T4 ligase. Namely, the pCMV-Luc comprises the CMVpromoter 61, luciferase gene 69, transcription termination signalsequence 65 and SV40ori sequence 67. The luciferase gene 69 andtranscription termination signal sequence 65 are incorporated at thedownstream of the CMV promoter 61. As mentioned above, the pCMV-Luc isfree from a replication origin-binding protein gene and a replicationorigin sequence, and thus functions as a non-self-amplified reportervector.

3. Expression of LT Protein in Host Cell

The pCMV-Luc-IRES-LT and pCMV-Luc are introduced into a human hepaticcancer cell (Huh-7). As mentioned above, the pCMV-Luc is introduced as avector for a negative control against the pCMV-Luc-IRES-LT.

Each plasmid vector was introduced into the cell by a lipofectionmethod. As an introducing reagent for the plasmid vector, Lipofectamine2000 (Life Technologies Corporation) was used. The operations for thelipofection were conducted according to the manual of the reagent.Briefly, a Lipofectamine/vector conjugate was formed by mixing 1.0 μl ofa cation lipid (Lipofectamine 2000) suspended in 50 μl of Opti-MEM and50 μl of Opti-MEM comprising 0.6 μg of the pCMV-Luc-IRES-LT or 0.4 μg ofthe pCMV-Luc and 0.2 μg of a DNA (pUC19). After formation of theLipofectamine/vector composite, 50 μl of this composite liquid was addedto a culture medium (Huh-7 (sown at 8.0×10⁴ cells/well)) that had beencultured overnight in advance in a culture plate (a 24-well plate) tointroduce the pCMV-Luc-IRES-LT into the cell.

At 48 hours after the lipofection, the culture medium was removed fromthe culture plate, and the cells in the culture medium were suspended ina phosphate buffer (PBS). After the suspension, the suspension liquidincluding the phosphate buffer and cells was centrifuged in a centrifugeat an angle velocity of 14,000 rpm over 5 minutes, and cell pellets werecollected from the suspension liquid. A cell lysate for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was added to thiscell pellets, and incubation was conducted over 5 minutes in boilingwater. Thereafter electrophoresis was conducted by SDS-PAGE of 8%. Afterthe electrophoresis, the protein in the gel was blotted on a PVDFmembrane by using a submarine-type blotting apparatus, and the PVDFmembrane was blocked with Block Ace (Dainippon Sumitomo Pharma). Afterthe blocking, the PVDF membrane was immersed in a primary antibodysolution (a mouse anti-LT monoclonal antibody (Clone 9E10, Sigma)diluted 500-fold) and reacted at a room temperature over 1 hour. ThePVDF membrane was then washed three times with tris-buffered saline(TBS).

After the washing, the amount of expression of the LT protein in thePVDF membrane was measured by using an ABC Kit (Alkaline PhosphataseUniversal, VECTASTAIN). The operations of the ABC Kit were conductedaccording to the manual of the selling manufacturer. The PVDF membrane(an enzyme-labeled secondary antibody) was color-developed with BCIP/NBT(KPL Inc.).

FIG. 6 is a drawing that shows the result of detection of the signal ofthe LT protein in the pCMV-Luc-IRES-LT and pCMV-Luc. As shown in FIG. 6,the expression of the LT protein was not detected in the pCMV-Luc. Onthe other hand, the expression of the LT protein was specificallydetected in the pCMV-Luc-IRES-LT. In the example of FIG. 6, themolecular weight of the signal of the LT protein was measured to beabout 82 kDa.

4. Amplification of Plasmid Vector

pCMV-Luc-IRES-LT and pCMV-Luc having a same copy number were introducedinto a Huh-7 cell according to a similar method to that in theabove-mentioned 3. At after 72 hours from the lipofection, a DNA wasextracted from the cell by using Dneasy Kit (Qiagen). The operations forextracting the DNA were conducted according to the manual of the kit.PCR was conducted by using 1.0 μl from 200 μl of the extracted DNAsolution as a template to amplify the partial base sequence of thepCMV-Luc-IRES-LT or pCMV-Luc. The base sequences of the primers used inthe PCR are shown below.

Forward primer: (SEQ ID NO: 10) 5′-CGACTGTGCCTTCTAGTTGCCAGCC-3′Reverse primer: (SEQ ID NO: 11) 5′-CCAGCATGCCTGCTATTGTCTTCCC-3′

The DNA solution after the PCR reaction was subjected to electrophoresiswith a 0.8% agarose gel. After the electrophoresis, the gel comprisingthe DNA solution was stained by ethidium bromide, and a picture of thestained gel was photographed. The staining intensity of a band drawn inthe ethidium bromide-stained picture was quantified by using imageanalysis software (Image J).

FIG. 7A and FIG. 7B show the result of the comparison of the copy numberof the pCMV-Luc-IRES-LT (the staining intensity of the band) against thepCMV-Luc at after 72 hours from the introduction of the plasmid vectorinto the cell. FIG. 7A shows the ethidium bromide-stained pictures ofthe pCMV-Luc and pCMV-Luc-IRES-LT. FIG. 7B shows a graph (a graph of astaining intensity ratio) for the comparison of the copy numbers of thepCMV-Luc and pCMV-Luc-IRES-LT. As shown in FIG. 7A, the luminance of thepicture is higher in the pCMV-Luc-IRES-LT than that in the pCMV-Luc.Therefore, it can be found from the ethidium bromide-stained picturethat the pCMV-Luc-IRES-LT provides a higher signal intensity than thepCMV-Luc does. Furthermore, as shown in FIG. 7B, the pCMV-Luc-IRES-LTprovides a higher signal intensity than that of the pCMV-Luc. It can befound from this graph that the copy number of the pCMV-Luc-IRES-LT hasbeen amplified to 4.2 times of the copy number of the pCMV-Luc.

5. Reporter Gene Assay-1-(Luciferase Assay)

The pCMV-Luc-IRES-LT and pCMV-Luc having a same copy number wereintroduced into a Huh-7 cell and a simian kidney cancer cell (CV-1),respectively, by a lipofection method in a manner similar to that of theabove-mentioned 3, and cultured on a culture plate. At 72 hours afterthe lipofection, the culture medium was removed from the culture plate,and each cell was washed twice with PBS, and a cell lysate that is anextraction reagent for extracting luciferase (a reporter protein) from acell (PicaGene Cell lysis buffer LCβ, TOYO B-Net Co., Ltd.) was addedthereto. The suspension liquid of the cell and cell lysate was incubatedat a room temperature for 15 minutes, and the suspension liquid was thencentrifuged by a centrifuge at an angle velocity of 15,000 rpm for 5minutes to remove a cell debris from the suspension liquid. Furthermore,a luciferase substrate solution (PicaGene LT2.0, TOYO B-Net Co., Ltd.)was added to the supernatant of the suspension liquid from which thecell debris had been removed, and the luminescence intensity of thesupernatant to which the luciferase substrate solution (luciferinsolution) that is a detection reagent had been added was then measuredby a luminometer (Mithras LB940, Berthold).

FIG. 8 is a drawing that shows the result of comparison of the signalintensities of the reporter proteins of the pCMV-Luc andpCMV-Luc-IRES-LT. As shown in FIG. 8, in the case when the host cell wasHuh-7, the signal intensity of the reporter protein of thepCMV-Luc-IRES-LT against the pCMV-Luc was about 5.7 times. In the casewhen the host cell was CV-1, the signal intensity of the reporterprotein of the pCMV-Luc-IRES-LT against the pCMV-Luc was about 19.8times. As mentioned above, the plasmid vector according to the presentembodiment can express up to about 19.8 times of a reporter protein ascompared to a non-amplified plasmid vector. Therefore, the reporter geneassay according to the present embodiment can detect the activity of agene promoter with higher sensitivity as compared to conventionalreporter gene assays.

6. Reporter Gene Assay-2-(Luciferase Assay)

The pCMV-Luc-IRES-LT, pCMV-Luc-IRES-LT-E107K, pCMV-Luc-IRES-LT-D402E andpCMV-Luc having a same copy number were introduced into a simian kidneycancer cell (CV-1) by a lipofection method in a manner similar to theabove-mentioned 3, and cultured on a culture plate. At 72 hours afterthe lipofection, the culture medium was removed from the culture plate,and each cell was washed twice with PBS, and a cell lysate that is anextraction reagent for extracting luciferase (a reporter protein) from acell (PicaGene Cell lysis buffer LCβ, TOYO B-Net Co., Ltd.) was addedthereto. The suspension liquid of the cell and cell lysate was incubatedat a room temperature for 15 minutes, and the suspension liquid was thencentrifuged by a centrifuge at an angle velocity of 15,000 rpm for 5minutes to remove a cell debris from the suspension liquid. Furthermore,a luciferase substrate solution (PicaGene LT2.0, TOYO B-Net Co., Ltd.)was added to the supernatant of the suspension liquid from which thecell debris had been removed, and the luminescence intensity of thesupernatant to which the luciferase substrate solution (luciferinsolution) that is a detection reagent had been added was then measuredby a luminometer (Mithras LB940, Berthold).

FIG. 9 is a drawing that shows the result of the comparison of thesignal intensities of the reporter protein of pCMV-Luc-IRES-LT,pCMV-Luc-IRES-LT-E107K and pCMV-Luc-IRES-LT-D402E against the pCMV-Luc.As shown in FIG. 10, the signal intensities of the reporter protein inthe pCMV-Luc-IRES-LT, pCMV-Luc-IRES-LT-E107K and pCMV-Luc-IRES-LT-D402Eagainst the pCMV-Luc were about 20 times, about 108 times and about 177times, respectively. As mentioned above, the plasmid vector according tothe present embodiment can express up to about 177 times of a reporterprotein as compared to a non-amplified plasmid vector, by using amutant-type LT gene. Therefore, the reporter gene assay according to thepresent embodiment can detect the activity of a gene promoter withhigher sensitivity as compared to conventional reporter gene assays.

[Effect]

As mentioned above, the plasmid vector 1 according to the presentembodiment has the gene promoter 10 to be detected, reporter gene 21,IRES 23, replication origin-binding protein gene 25, transcriptiontermination signal sequence 27 and replication origin sequence 30. Thereporter gene 21, IRES 23, replication origin-binding protein gene 25and transcription termination signal sequence 27 are disposed at thedownstream of the gene promoter 10. The IRES 23 links the reporter gene21 to the replication origin-binding protein gene 25. Therefore, in thecase when the plasmid vector 1 is introduced into a host cell thatsatisfies the activation condition of the gene promoter 10, a reporterprotein is synthesized in the individual plasmid vector 1 while theplasmid vector 1 repeats replication in accordance with the activationof the gene promoter 10. Therefore, the plasmid vector 1 according tothe present embodiment can increase more the amount of expression of areporter protein that is synthesized due to the activity of the genepromoter 10, i.e., the amount of expression of the measured signal, ascompared to a conventional non-amplified reporter vector. Furthermore,since the plasmid vector 1 according to the present embodiment utilizesa plasmid, it has no risk of infection and the like and is handledconveniently as compared to a vector utilizing a virus.

In the reporter gene assay according to the present embodiment, a hostcell is cultured under a condition in which the plasmid vector 1 can beamplified in accordance with the activity of the gene promoter 10included in the plasmid vector 1, and the amount of expression of thereporter gene 21 included in the plasmid vector 1 in the cultured hostcell is measured. Therefore, the reporter gene assay according to thepresent embodiment can detect the activity of a gene promoter withhigher sensitivity as compared to a conventional reporter gene assaythat utilizes a non-amplified reporter vector.

Furthermore, the assay kit according to the present embodiment comprisesthe plasmid vector 1, and a reagent for detecting the expression of thereporter gene 21 included in the plasmid vector 1 in a host cell.Therefore, the assay kit according to the present embodiment can detectthe activity of a gene promoter with higher sensitivity as compared to aconventional assay kit that utilizes a non-amplified reporter vector.

For example, in the case when the gene promoter 10 that is specificallyactivated in a cell in a pre-disease state at an early stage of adisease is incorporated in the plasmid vector 1, the amount ofexpression of the reporter protein per the unit number of the cell inthis pre-disease state can be increased more significantly than before.By the increase in the amount of expression, the expression of thereporter protein can be detected easily, which consequently enables easydetection of the activity of the gene promoter 10. Thus, according tothe present embodiment, the change in an infinitesimal quantity of alesion cell can be detected with high sensitivity.

Thus, the present embodiment can provide a plasmid vector, a method fordetecting a gene promoter and an assay kit, by which the activity of agene promoter can be detected with high sensitivity.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the inventions. Indeed, the novel embodiments described hereinmay be embodied in a variety of other forms; furthermore, variousomissions, substitutions and changes in the form of the embodimentsdescribed herein may be made without departing from the spirit of theinventions. The accompanying claims and their equivalents are intendedto cover such forms or modifications as would fall within the scope andspirit of the inventions.

What is claimed is:
 1. A method for detecting the activity of a genepromoter, comprising: introducing a circular plasmid vector into a cell;culturing the cell under a condition where the cell divides andproliferates; determining expression of a reporter gene in the plasmidvector in the cultured cell, wherein the reporter gene encodes areporter protein and the reporter gene is functionally linked downstreamof the gene promoter; and determining the gene promoter activity;wherein the gene promoter is activated in a host cell that satisfies anactivation condition of the gene promoter, wherein the circular plasmidvector comprises in addition to the gene promoter and the reporter gene:an internal ribosome entry site that is linked downstream of thereporter gene, a base sequence that encodes a replication origin-bindingprotein, the base sequence linked downstream of the internal ribosomeentry site, wherein the base sequence is a large T-antigen gene ofSimian Virus 40 and has the base sequence of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 3, or SEQ ID NO: 4, a transcription termination signalsequence for encoding a signal for terminating the transcription of thefirst base sequence and second base sequence, and a replication originsequence that is recognized by the replication origin-binding protein,linked downstream of the transcription termination signal sequence,wherein the replication origin sequence is a replication origin sequenceof Simian Virus
 40. 2. The detection method of claim 1, wherein the cellis a cell derived from a human or a simian.
 3. The detection method ofclaim 1, wherein the reporter gene is a luciferase gene, aβ-galactosidase gene, a blue fluorescent protein gene, a greenfluorescent protein gene or a red fluorescent protein gene, and themeasuring comprises measuring the amount of light emission or the amountof fluorescence of a translation product of the reporter gene as theamount of expression by utilizing an optical detection apparatus.
 4. Thedetection method of claim 1, wherein the gene promoter can be activatedunder a condition in which the cell divides and proliferates.
 5. Thedetection method of claim 1, wherein the gene promoter is a genepromoter that is activated at an early stage of a disease, or a genepromoter that is activated in response to an environmental stimulus. 6.An assay kit comprising: a plasmid vector, and a reagent for detectingthe expression of a gene that encodes a reporter protein included in theplasmid vector in a cell, wherein the circular plasmid vector comprises:a gene promoter to be detected, which is activated in a host cell thatsatisfies an activation condition of the gene promoter, a first basesequence of a first gene that encodes the reporter protein forvisualizing the activity of the gene promoter, the first base sequencefunctionally linked downstream of the gene promoter, an internalribosome entry site that is linked downstream of the first sequence, asecond base sequence of a second gene that encodes a replicationorigin-binding protein, the second base sequence linked downstream ofthe internal ribosome entry site, wherein the second base sequence is alarge T-antigen gene of Simian Virus 40 and has the base sequence of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, a transcriptiontermination signal sequence for encoding a signal for terminating thetranscription of the first base sequence and second base sequence, and areplication origin sequence that is recognized by the replicationorigin-binding protein, linked downstream of the transcriptiontermination signal sequence, wherein the replication origin sequence isa replication origin sequence of Simian Virus
 40. 7. The assay kit ofclaim 6, which further comprises a reagent for introducing the plasmidvector into the cell.
 8. The assay kit of claim 6, which furthercomprises a reagent for extracting the reporter protein from the cell.9. The assay kit of claim 6, wherein the first gene is a luciferasegene, a β-galactosidase gene, a nitric monoxide synthase gene, axanthine oxidase gene, a blue fluorescent protein gene, a greenfluorescent protein gene, a red fluorescent protein gene or a heavymetal-binding protein gene.
 10. The assay kit of claim 6, wherein thegene promoter is a gene promoter that is activated at an early stage ofa disease, or a gene promoter that is activated in response to anenvironmental stimulus.
 11. A circular plasmid vector comprises: a genepromoter to be detected, which is activated in a host cell thatsatisfies an activation condition of the gene promoter, a first basesequence of a first gene that encodes a reporter protein for visualizingthe activity of the gene promoter, the first base sequence functionallylinked downstream of the gene promoter, an internal ribosome entry sitethat is linked downstream of the first base sequence, a second basesequence of a second gene that encodes a replication origin-bindingprotein, the second base sequence linked downstream of the internalribosome entry site, wherein the second base sequence is a largeT-antigen gene of Simian Virus 40 and has the base sequence of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, a transcriptiontermination signal sequence for encoding a signal for terminating thetranscription of the first base sequence and the second base sequence,and a replication origin sequence that is recognized by the replicationorigin-binding protein, linked downstream of the transcriptiontermination signal sequence, wherein the replication origin sequence isa replication origin sequence of Simian Virus
 40. 12. The plasmid vectorof claim 11, wherein the first gene is a luciferase gene, aβ-galactosidase gene, a nitric monoxide synthase gene, a xanthineoxidase gene, a blue fluorescent protein gene, a green fluorescentprotein gene, a red fluorescent protein gene or a heavy metal-bindingprotein gene.
 13. The plasmid vector of claim 11, wherein the genepromoter is a gene promoter that is activated at an early stage of adisease, or a gene promoter that is activated in response to anenvironmental stimulus.